Bayer Martin J, Reese Christoph, Buhler Susanne, Peters Christopher, Mayer Andreas
Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, 72076 Tübingen, Germany.
J Cell Biol. 2003 Jul 21;162(2):211-22. doi: 10.1083/jcb.200212004.
Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V0 sectors in vacuole fusion. V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse. The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release. These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.
膜融合的孔模型假定，整合膜蛋白圆柱体在孔亚基构象重排后可启动融合孔。在酵母液泡融合过程中，V-ATPase V0扇区包含膜整合蛋白脂质亚基的中央圆柱体，它们相互结合形成一个反式复合体，该复合体可能类似于某些孔模型中假定的中间体。我们测试了V0扇区在液泡融合中的作用。通过突变和抑制性抗体的不同作用，可以在实验上分离V0在融合和质子转运中的功能。V0亚基Vph1p的失活在与质子梯度无关的终末反应阶段阻断融合。Deltavph1突变体能够对接并进行反式SNARE配对，随后释放腔内Ca2+，但它们不会融合。Ca2+释放通道似乎与V0紧密偶联，因为抗体使Vph1p失活会阻断Ca2+释放。仅在一个融合伙伴上缺失Vph1就足以严重降低融合活性。对Vph1p的功能需求与V0反式复合体的形成相关，因为两者都发生在对接和Ca2+释放之后。这些观察结果表明V0是液泡融合中反式SNARE配对下游的关键因素。
